Protocols

All the protocols we followed in our project.

Cloning protocols

Here are the protocols we followed for cloning our linkers into the existing R body plasmids. We used these protocols to perform the experiments described in the overview.

PCR protocol

Materials

  • Microfuge tubes
  • PCR tubes
  • PCR thermocycler
  • Micropipettes (P20, P200, P1000 are all likely useful here, depending on the number of PCR reactions to be carried out)
  • Milli-Q water (purified water)
  • 5x Phusion buffer
  • Phusion enzyme
  • Forward primer (10 µM)
  • Reverse primer (10 µM)
  • Template (50 ng/µL)
  • dNTPs

Instructions

  1. Make up Master Mix on ice, as in the table below (excluding the primers). Make two extra reactions worth than required for the number of samples required for amplification, as a buffer against losing Master Mix due to mistakes. For performing PCR with DMSO, replace the purified water with DMSO (e.g. for 5% DMSO, use 2.5 µL DMSO and 27.5 µL purified water per reaction). Mix the Master Mix excluding the primers in a microfuge tube first to reduce the chance of non-specific binding. Add the primers to labelled PCR tubes.
Ingredient Amount per reaction (µL)
5x Phusion buffer 10
dNTPs 4
Template 0.5
Purified water (Milli-Q) 30
Phusion polymerase 0.5
F primer 2.5
R primer 2.5
Total reaction volume: 50

  1. Add 45 µL of Master Mix to each PCR tubes with primers. Keep the whole solutions on ice for as long as possible.

  2. Place the PCR solutions in the thermocycler, and run the program below:

Step Time (mm:ss) Temperature (°C) Repeats
Initial denaturation 02:00 98.0 1
Denaturation 00:20 98.0 35
Annealing 00:20 Tm
Extension Fragment length (rounded up to next kbp) × 00:30 72.0
Final extension 2 × fragment length (rounded up to next kbp) × 00:30 72.0 1
Hold - (until ready to retrieve sample) 4.0 -